This project will study the biosynthesis of human von Willebrand (vW) protein cultured endothelial cells. The plan of study is based upon two new observations that were recently made by Dr. Wagner. First, fluorescent light microscopy of permeabilized endothelial cells demonstrates that the vW protein appears to concentrate in cytoplasmic vesicles, the structural features of which we interpret to represent the Weibel-Palade bodies. Second, we have identified a 260 kd polypeptide chain within the endothelial cell which reacts with monovalent rabbit anti-human vW protein antiserum and which appears to represent a precursor form of the 200 kd reduced chain subunit of vW protein. We propose to pursue these observations by the following experimental approaches. The subcellular localization of the vW protein will be elucidated, primarily by immunoelectron microscopy and by subcellular fractionation of metabolically-labeled cells. The sequence of molecular events which occurs within the cells during biosynthesis will be approached using pulse-chase experiments, with emphasis on studies of protomeric precursor forms, mode of disulfide bond and multimer formation, and carbohydrate addition and processing. The observations on biosynthesis will be correlated with those on anatomic subcellular localization, to reveal insights on the mode jin which vW protein is handled by endothelial cells. Last, the steady-state secretion and extracellular fate of vW protein will be followed, with an attempt to induce secretion by appropriate pharmacologic stimuli. The information provided by these studies of biosynthesis and subcellular handling of vW protein by endothelial cells will provide new insights into mechanisms for hemostasis and thrombosis, as well as a better understanding of the molecular or cellular derangements that underlie von Willebrand's disease.